Cycloserine and production thereof



Dec. l, 1956 G. M. sHuLn. Erm.

CYCLOSERENE AND PRODUCTION THEREOF Filed March 5, 1952 Sm Ss EN. Sm. .5....

12o z. wmmmaz u??v 5L A MLNY. TUE N NH..L R ,STN M .w1 c o NMR w Ta R @LNE www @JVM E L A United States Patent CYCLGSERINE AND PRDUCTICN MREGF lae. This strainrwhich we have isolated'fr'om soil, and which We designate as culture number 8197-20, appears to be very similar in cultural characteristics to a strain of S. lavendnlae obtained from Di'. Selman-Waksman,

Pigment*Light'-grayishr brown 'to dark brown'.

Remarks--No coagulation; yery slight peptonization o millr.`

See footnote at end of table Light to'fairly darkbrown.

N o coagulation; very slight peptonizaizion.-`

5k with minor exceptions. A culture of the living micro- Gilbert M. Shull, Roslyn Heights, N. Y., John B. Routen, organism has been deposited with and is available from Tenaily, N. J., and Alexander C. Finlay, Flushing, the'Ameriean TypeCulture Collection; it has been desig- N. Y., assignors .to Chas. Pizera Co., Inc., Brooklyn, Dated as ATCC 11924 N- Y-s a corporation 0f Deiawa The new strain was planted on two occasions onto the :er Application March 5, 1952, Serial N0 274,924 10 standard medla used for identication of actinomycetes. AtV the Sametime a culture sent'to us b Dr. Waksman s claims. (ci. 26o-307) S l d l .y .u as aven u ae, and which has been givenour number ,it F; D. 1073, was planted on the same lots of media.

Listed below are the cultural characteristi Thls Inventum 1s Concerned Wlth a new and useful 15 strains as well as the descri tive hrases ass iifbtlileste tiiio antibiotic called cycloserine. It is also concerned with s ecies b Waksman a d Hp B ,C A; o1 the production of this antibiotic, particularly by fermenat B .nl em-l? egey? aupa 9, tation, and its recovery and concentration from crude er'mma we ateno ogy e m09 y P1: 944; solutions, including fermentation broths. The invention Readmgs are based- 0D a total 0f 11H16 tubes or Petri embraces cycloserine in dilute solutions, as crude concen- 20 dishes 0f each medium for each CUll-ll'e- (The C0101'S, trates, and in pued form where R 1s written, are those of Robert Ridgway, Color This new antibiotic is formed during the cultivation Saldafds and Nomenclature, 1912-) 0111 Culture iS S0 under controlled conditions of a novel strain of the Similar t0 Di. Waksmans strain that we consider it to known species of microorganismi, Szreptomyces lavendubelong to the same species, S. lavendulae.

Culture Medium f Our strain 8197-20 Waksmans strain, our F. D. 1073 Waksmans and Henricis description l Growth-poor to good Poor to good Spores-good sporulation, near 'pale` sporulation poor to good, near pale vinaceous drab (R vinaceous'drab (R). unliil'ili f igmmi-lmiem-""iiii Alorolnemiicmiiin 1 A omar so ony a to sig y e ef' o any.. a o s ig y e evate surspirals close.` C onidia'oyal,-1.0 to 1.2 Ghcgseaspamgme agar vated, ,surface smooth to slightly face slightly wrinkled; edge smooth; by 1.6 to 2.0 microns,

p a es' wrinkled; edgel smooth; reverse reverseVA creamy to light brown. creamy color. spores'in chairs,V Sporesin mostly straight orslightly chains mostly.straight or slightly curved chairs; very few rather loose curved; very fewloose =pirals.f spirals; spores short-cylindrical, Sporasf short-cylindrical, 0.65-i.0" 0.65-1x1.3-2.0;i. ri-2.0,..

rowth-Moderate at 18C.` Moderate to good i spores-None; colony waxy and=light None, colony waxy, brown to tan. Creamy to brownish surface growth. K brown to near olive blu (R). Lqueied. Gelatin plates Soluble l Pigment-Dark brown Dark brown Rmirks-Moderate -to good lique Very poor liqnefaction ac ion; Growth-Moderate Moderate to good spores-Mostly none, colony waxy, None; colony waxy, creamy to light Apale tan; occasional whiteaeriahbrownish. Glucose agar mycelium. y Y

' Soluble Pigment-Mediumto dark brown. Medium to dark brownu, Growth-Poor, thin Poor, thin Spares-sporulation moderate, white From none to'slight sporulation of arlill mycellium with pale'lrrolwrislA smoke gray (R) to pale pink color. p' to pao vinaceous a q Calcium malate spores L olnble a Pigment-None ,Remarks-Reverse white GrOwth P0m- Restrizttd, glsteningyf transparent gro p Spores-'-tloorl to good" sporulation, Pfgor spotrulation iroimpaleupnkish white o pa e gray spores; rown o near smo e gray Starch plates Som 1e Pigment-None None..` k Rilalmrkls-Reverse"whiteyalmost no Reverse'white; almost no hydrolisis... Hydrolyzed.

y oysis. Growthmpoorfhm 4 Poor-,thru v Y Ehm-spreading 'colorlessg`rc:wt'tx'. Spores-Spoi'ulation'good, near pale Ysporulation poor-torgood, very pale Aerial mycelium 'cotton'y, whitebe'- vinaceous fawn to vinaceous bui pink to near avellaneous (R). coming vinous-lavender. Synthetic agar (R): f

Soluble Pigment-Nona None Remarks-Reversewhite B everse white; Cellulose strips Growth-None 0ne-.f

Growth-Moderate: Moderate (On litmus milla),V Spores-Brownish rngintube Ciagn 'colored to Vbrownish ring in Cream colored ring.

e. skimmed .u YV Soluble No coagulation, l?eptivinireci,r`

Culture Medium Our strain 8197- Waksmans strain, our F. D. 1073 Waksmans and Henricis description l glo rluction No reduction Nitrites produced. o u e. Dextmse mtrate broth Pigment-Light brown Light to medium brown. Remarks-Reverse light brown- Reverse light brown.

Nutrient agar Potato plugs Growth-Moderata Sp orcs-Very few; colony surface white Soluble.

Poor to moderate None; colony waxy, creamy to grayish brown.

Pigment-Light brown Remarks-Creamy to light tan Growth--Good Spores-Sporulation moderate to good; light vinaceous fawn to pallid vinaceous drab to avellaneous and light cinnamon drab (R), with some waxy areas. Growth-Good.-.. l spores-Good sporulation, near, light cinnamon drab to avellaneous (R).

mouse gray (R) with most of colony waxy to good sporulation of pallid mouse gray to mouse gray (R) color.

Good.- Moderate to good sporulation, near light cinnamon drab to vinaceous owisli growth.

Pigment-Light to medium brown--- RemarksfReverse light to medium brown.

Light to medium brown Reverse light to medium brown l V1.1i:i Bergeys Manual.

Heretofore strains of S( luvendulae have been reported to produce two different antibiotics, lavendulin (Kelner et al., I. Bact., vol. 51, p. 591 (1946)) and streptothricin (Waksmari et al., Soil Science, vol. 54, p. 281-296 (1942)). 'Y Cycloserine is wholly distinct from either of "these, as well as from other known antibiotics. The notably low toxicity of cycloserine makes it of special value therapeutically, in contrast to lavendulin and streptothricin which havek particularly high toxicities.

Itis to be understood that for the'production of cyclo serine we do'not wish 'to limit ourselves to the particular' isolate here described, as-variations may occur in the cultural characteristics of this strain without affecting production of the antibiotic. We especially wish to include in the term strain No.'ATCC 11924 organisms which are mutants obtained by mutating agents such as X-radiation, ultraviolet radiation, nitrogen mustards and so forth.

Cycloserine shows considerable activity against a number of different gram-negative and gram-positive microorganisms, and notably against the mycobacteria. It is effective in human therapy for bacterial infections and tuberculosis. ,With certain other microorganisms, such as Manilla albicans and T richophyton gypseum, its acaureus to which various amounts of the given sample have been added. (See McMahon, I. Biol. Chem., vol. 153, p. 249-258, (1949).) A third testutilizes a standard culture of E. coli, and this method forms the present preferred basis for assigning potencies to the various preparations of cycloserine.y 'l`hesepotenciesY are dened in terms of E. coli dilution units (C. D. U.) per milligram. By this we mean the maximum volume of nutrient broth in milliliters to which one milligram of the antibiotic preparation (which may be ofvarying degrees ofpurity) may be diluted and, when inoculated with a 10"B dilution of an 18-hour culture of E. coli grown under standard conditions, still show no bacterial growth at the end of 18 hours incubation at 37 C. l

The following table shows the antibiotic spectrum of a crude preparation of the newk composition against a number of diierent microorganisms. The sample tested tivity has not yet been proven in therapy.V It has unusually low toxicity as an advantageous accompaniment to its generally high activity. Forinstance, 100 mg. of the crude preparation described in the table below may be injected intravenously Vinto 1Z0-gram mice without causingdeath. Morehighly purified cycloserine is natu-UY The zone'of inhibition formed upon addpendent upon the pH of the test solution of cycloserine,

the highest activity being obtained at a pH of about 2. Y 4 Mmsmegmm A, second method for testing the new antibiotic, not sensitive to pH. changes, is by measuring the extent of turbidity in standard liquid cultures of Staphylococcus had a potency of 200 C.'D.V U./mg. Purer samples demonstrate higher activity.r The figures in the table indicate the concentrations in micrograms of the cycloserine preparation required per milliliter of nutrient agar, to prevent bacterial growth on the agar; the symbol v-lindicates growth, and no growth.

Concentration' Mioroorganim A. aeroge'lies 1 Proteus sp.--" Pseudomonas s 1 i i+1 i H l i+1 l i l i 1+|++| i i 1 I+II il l l i+|-|-++|+++ ++|++i`|+++++++++++++ a 1 Aureomycln-resistant strain. i streptomycin-resistant strain.

'Whenz a sample of crystallinecycloserine is i4usedfto' determine fthe, activity ,against theseYrnicroer-"g'anisms, the,"

followingx resultsV are obtained. This material.: has a potencyv'of about 1300 lunits per milligram.-

1 Aureomsvcinlresistant strain. 2 streptomycin-resistant strain.

Cycloserine has been compared to other antibiotics anclfound to be a distinctly different compound. For instance, the new antibiotic, when tested by paper chromatography vwith various known materials using sev' eral solvent systems, formed distinct, compactzones on Sheets f. filter paper..

, Thisinvention embraces a process for` producing Icycloserine vby' cultivating the new'strain-No. ATCC l1924of the microorganisrn Streptomyces lavendzzlae in-an aqueous, nutrientcontaining solution under aerobic conditions until" substantial antibiotic activity* has been imparted -to the solution.r` Suchcultivation is conducted preferably at a ternperatureof fromn about 25 toabout 30 C. vand under submerged 'conditions of agitationand aeration; for a period generally of fronlabout two to four days. Nutrient materials that'may be used include an organic nitrpgerh source,i.such f as.:soybean i meal, 1 cottonseed'rmeal, alfalfa meal, hydrolyzates.- .of .-'casein.,and. other proteins, etc` .a,sourcel of growth substance, such as distillers solubles, yeast extract, or a commercially availablevitamin concentrate (BY500); mineral salts like sodium ,chloride and potassium phosphate, but especially sodium, nitrate; a'bufl'ering'agent'like calcium carbonate; andj an oil to help preventfoaming, e. g. soybean oil, lard oil, a silicone or% .otherz suitable A. material.Y Particularly beneficial, V as nutrientsarecarbohydrate sources, l such as sugars, starch, glycerineI .and Ythe like. After growth hasbeen completed, the mycelium=is separated, from` the broth -now containing thefantibiotic, and cyeloserine is desirably recoveredrom theabroth; byv suitablemeans which .will bev described below'.A

Inoculumfor` thisermentation may be obtained-by ernployinga growthffromyslantsor Roux bottles inoculated withfounstrain of S. lavendulae. Solid mediay for the initial-,growth arecomrnonly beef lactose and VEmersons agar,. and.this growth is used to inoculate either shaken flasks or,submerged inoculum tanks. Alternatively, 4the inoculum. tanks are seededfrom theY shaken asks.- kThe time requiredfor optimumelaboration of cycloserine is generally -at least about two days but notcmore than about ve days..,When the material lisproduced; byl growth in shaken asks, the time is generally longer than in stirred, aerated tanks. At all times aeration is maintained in thetanks by blowing sterile air through a sparger at 'the'jrate-of 1A. to2 volumes of free airper volume of broth per-minute. Certain antifoaming agents, such as vegetableioranimal oils or a silicone,.help' to .prevent excessive foaming. The broth is agitated at a speed depending upon?v -thefparticulartype of agitatorused in the tank, and

completelylaseptic conditions aref maintained:y throughs out the growth..

The crude! broth obtainedv after filtrationv of. the mycelium from `the new.. strain of Si vlavenalulae has utility in itself. However, we preferto purify the cycloserineat least partially before using it therapeutically. Purification may bebroughtabout by certain yparticularly ecient techniques. Thus, the antibiotic may be adsorbedzfrom solution on strongly acidic cation-exchange resins, especially those containing sulfonic acid groupsas the active -adsorbing radicals. Of particular utilityy are ,the resins known in the trade'as Amberlites, notably'fAmberlite IR 120i Adsorption is best conducted with'these at from about pH.3.0'to about. pH 7.0the higher range being more favorable. Other strongly acidic ionexchange resins, such as AmberliteIR 'or Amberline 1R11, may be used, but their capacity is less thanthat of Amberlite IR 120. Other .sulfonic acid type resins, such: as Amberlite IR 105, PermutitxQj Ionac (Tf-200,. or Dowex 30 or "Dowex 50 are alsovaluable.- These materials' Vare generally phenol-formaldehyde resins y:containing sulfonic Aacid. groups introduced by addinga sulte during formation of the resin; or they are sulfonated hydrocarbon polymers or polymers prepared from-sulfonated phenols and formaldehyde.

The adsorbate rof cycloserine on such ion-exchange resinshas-.utility of itself, e. g.V it may be us'ed'as ka source ofthe antibiotic in'animal. nutrition or in treating specific diseases of animals or human beings. Nevertheless, we prefer to elute the antibiotic from the resin and use: a purified vcor'icentrate obtained therefrom. This may be still further purified, as described in detailbelow. For elution of the antibiotic from the resin, it has been found that dilute alkaline `solutions are particularly useful. For instance, ammonium hydroxide. at-a concentration of about 0.5 to about 3% -by weight in water is quite useful for displacing the antibiotic from the resin. This may be done in a fractional manner so thatV a product of particularly high activity is obtained. After adsorption of the antibiotic on .the resin, the resin may be washed withasmall volume of water .to remove' residualy inactive material, and the dilute basic solution, in particularammonium hydroxide, may .then be slowly'run through the resin. Although we nd it quite useful to employ the resin in a column or tube, adsorption. and elution may be Acarried out alternativelyv in batchwise fashion. Other bases, such` as sodium or potassium hydroxide or strong organic bases, can readily be substituted for the ammonium compound.

When fractionally eluting` cycloserine from resin` adsorbate, the rst eluate from the resin bed has a strongly acidic reaction. As more and more of the dilute baseris passed throughthe column of pH of the eiuent gradually rises. Small amounts of active material areobtained in the first effluent, but not until the pH. reaches about 6 is the major portion of the antibiotic recovered. At a pH of from about 6 to 6.5 much of the. cycloserine is removed. However, the optimum pH may varyk considerably with the batch of antibiotic used. In some 'cases a pH of as high as l0 is required to .remove most of the antibiotic. The highly active fractionofe'luent may be separated from the forerun and afterrunto yield a dried product of particularly high purity. Material obtained by preliminary purification of the'fbrothwith activated carbon at a basic pH and then at an acidic pH (as hereinafter discussed), followed by :adsorption -on Amberlite IR and fractional elution with ammonium hydroxide, will yield a solid product having a cycloserine potency of about 200 C. D. U./ mg. .or greater. Such `product may contain 10% or less ofash,.whicl1fcan be almost completely removedby passing a solution of this. product over a columnof a strongly basic type anion-exchange resin. Some loss of potency. is encountered in such a purification, however, :and a preferred method is chromatography on columns of alumina,.silca gel or vother suitable metallic oxide. Thus, if a'concentrated solution of the antibiotic in water orvin water containing methanol is passed over a column of activated alumina, .the antibiotic is adsorbed thereon. By passing 'aqueous methanol over such'a column, it is possible to obtain fractions with higher activity. We have found that cycloserine is not adsorbed readily on activated carbon at either an alkaline or an acid pH. This peculiar property of the antibiotic We use to advan- -tage by lselectively adsorbing a variety of impurities with carbon from the fermentation broth or from partially puried concentrates. For instance, ltered `cycloserine broth may be made basic and many impurities removed 'by activated carbon, such as Norit A or Nuchar. The solution obtained after removal of this carbon may be made acid and other impurities adsorbed by a second carbon treatment. When these two lsteps are carried out, llittle of the antibiotic is removed but a practically colorless solution is obtained. Such solution may be simply dried, or Vthe antibiotic may be adsorbed therefrom on vthe aforementioned Amberlite IR 120 or other suitable strongly acidic resin for further purification. Rather -than employing the carbon treatment before the adsorption with ion-exchange resin, the carbon may be used after elution of the antibiotic from the resin adsorbate. Various purification steps may be combined in order to obtain highly puried cycloserine and, in fact, the compound may be obtained in crystalline form by such procedures. For instance, fermentation broth may be `ltered at an acidic pH and treated with activated carbon to remove certain impurities. The antibiotic may then be adsorbed from the acid solution on a strongly acidic ion-exchange resin, such as Amberlite IR 120. VElutio'n is accomplished as described above with dilute ammonium hydroxide. The fractions containing the most highly potent material are collected and dried, preferably from the frozen stateunder vacuum. The dry material may then be extracted with methanol and the ltered extract may be treated with an excess of acetone to pre- `cipitatecertain impurities. On concentration of the liltrate, impurities separate. When a small volume is reached, the crystalline antibiotic nally separates. It maybe recrystallized from hot methanol, using carbon and a lteraid, particularly such materials as Super Filtrol, to remove such impurities. On cooling, the crystalline antibiotic gradually separates. An alternative method for crystallizing or recrystallizing the .antibiotic is to dissolve the purified material in a minimum amount of water and then add ethanol. It should be pointed out that prolonged heating of cycloserine, either in solvents orwater, 'tends to inactivate the material. Therefore, in. purification procedures, the length of time during whichV solutions of the antibiotic are heated should be kept to a minimum.

A distinct and unusual property of this new antibiotic lcycloserine is its non-extractability from fermentation broth either at acid, neutral or alkaline pHs with a variety ofk conventional organic solvents including butanol, methylisobutyl ketone, ether, ethylacetate, chloroform and benzene. It will be noted that these solvents represent manyditerent types of organic compounds: alcohols, ketones, -ethers, esters, chlorinated hydrocarbons and aromatic hydrocarbons. However, surprisingly enough, the addition of a strong organic acid like paratoluenesulfonic acid to cycloserine solutions implements extraction of the antibiotic into a water-immiscible alcohol-type solvent, such as butanol or benzyl alcohol. vOther strong organic acids may naturally be used in place siderable stability both kto heat .and pH changeswheny dissolved in water. Various crude :preparations ,of the antibiotic give strongly positive ferrie chloride tests.A Concentrates show little'absorption in the ultraviolet range .of the spectrum: Cycloserine itself, however, has a characteristic ultravioletrabsorption at a wavelength" of 225 microns.

. Samples of the crystalline `cycloserine-have been analyzed. The following is the elemental analysis of cycloserine calculated from the Vempirical formula CsIIsNaOs:V

It has beenrfound that analyses of the nitrogen content of this compound are particularly difficult and, unless care is exercised, low values are encountered. The opti-l cal rotation of the crystalline cycloserine, when dissolved in 90% methanol, is [a1D25=-l53.7. 'Ihe crystallinel compound is slightly soluble in butanol and acetone, somewhat more soluble in methanol, very slightly soluble or insoluble'in most other organic solvents, e. g. benzene, chloroform and ether, but very highly soluble in water. It should be noted that the presence of impurities tends to raise the solubility of the antibiotic appreciably.V 'The crystalline antibiotic has an apparent melting point of l55156 C. p F

The infrared absorption `spectrum of the crystalline compound was determined on a mineral oil mull. Among the characteristic frequencies are the following (in reciprocal centimeters): 1626, 1602, 1580, 1557, 1534, 1408, 1402, 1266, 1227, 1168, 1141, 1117, 1064, 938, 916, 892, `880, and 830. The infrared absorptionspectrum is shown in Figure I. v

The following examples are given as illustrations of the mannerin which cycloserine may be formed, recovered,"

concentrated, purified and obtained in therapeutically useful form. The examples given are merely illustrative and are not to be construed as limiting this invention.

Example I A fermentation medium containing the following `pto- -f portions of ingredients was prepared: Y Parts by weight This material was made up with distilled water to provide 41 grams per liter, and the mixture was adjusted to pH 7.0 with potassium hydroxidesolution. To the mixture were added per liter 5.0 grams of calcium carbonate and 7.5 ml. of soybean oil. Two thousand milliliter portions of this medium were then added to fermentation vessels, equipped with stirrers and aeration spargers, and sterilized at 121 C. for 60 minutes. After cooling the flasks were inocculated with a suspension of our new strain No'. ATCC 11924 of S. lavendulae, obtained from the surface of agar slants. The flasks were stirred for four Vdaysat 28 C. at approximately 1700 R. P. M. At the end of this period the broth was found `to contain cyclosterine in the amount of about 250 C. D. U./ml.' of Y broth. The mycelium was separated from the broth by filtration. The broth had a pH of about 7.5. Tests showed it tobe highly active against a variety of microorganisms.

Example Il One liter of cycloserine fermentation ybroth obtained as in Example I was adjusted to pH V10 with dilute sodium hydroxide and stirred with 10 grams of activated carbon for 30 minutes. The carbon was removed with a dia- .tomaceous earth lteraid and thefermentationbroth was adjusted to pH 2 with dilute sulfuric acid. A further portion of activated carbon was added to the broth, the mixture was stirred for 30 minutes, and the activated carbon was again removed by filtration and washed with a small volume of water. There was little loss in cycloserine activity and the solution obtained was practically colorless. The partially purified acid solution of cycloserine was then adsorbed on a column of Amberlite IR 120 in the hydrogen cycle. The column was washed with a small volume of water and then the antibiotic was eluted fractionally by means of a solution containing one milliliter of concentrated ammonium hydroxide per 100 milliliters of solution. Little activity was removed from the column until the eluent reached a pH of 6.1. The fraction obtained with the eiuent at a pH of 6.1 to 6.4 was recovered and dried from the frozen state. The product consisted of a ne, pale pink powder having a cycloserine activity of 200 C. D. U./mg. It was most suitable for use in therapy because of its low toxicity and considerable antibiotic activity.

A portion of this concentrate was dissolved in a small volume of 50% methanol and applied to a column of activated alumina which had been washed with 50% aqueous methanol. Further 50% methanol was passed through the column to develop and purify the antibiotic fractions. As the solution passed out of the column, it was divided into a number of fractions of equal volume. By removing the methanol under vacuum and drying from the frozen state, material of high cycloserine potency and having substantially no color was obtained.

Example III Fermentation broth prepared by growing Streptomyces lavendulae ATCC 11924 in a nutrient medium was found to have a pH of 7-8. Sulfuric acid was added to the whole fermentation broth with stirring and, when a pH of 2 was reached, the mycelium was filtered. Activated carbon was added to the ltrate at a concentration of about 2 percent. After stirring at room temperature for half an hour, the carbon was removed and between 10 and percent of the biological activity of the solution was lost, but considerable impurities and colored materials were removed. The antibiotic was adsorbed on Arnberlite IR 120 ion-exchange resin by passing the solution through a column of this material. The column was washed with a small volume of water and the product was eluted with dilute ammonium hydroxide. The red solution which was obtained from the column was partially concentrated under vacuum at room temperature or slightly lower and the concentrate was frozen and dried under vacuum. The dry product was extracted several times with methanol. The methanol extracts were filtered and iive volumes of acetone were added to the filtrate. The precipitated impurities were removed by filtration and the solution was concentrated to about 1/30 to 1/40 volume. Certain impurities separated during this concentration. These were filtered and the ltrate was again concentrated to about lo its volume. A crude, crystalline antibiotic product separated during this concentration. This material is tan or reddish in color.

The crude, crystalline product obtained by the procedure described above may be used for certain therapeutic purposes. However, it may be preferred to purify the material further. This may be accomplished by recrystallization from hot methanol. Activated carbon and a iilteraid, such as, Super Filtrol, may be used to remove the various colored impurities which adhere to the product. On cooling the filtered methanol solution, white crystals slowly separated. A further crop of material was obtained by concentration and cooling. Alternatively the crude, crystalline antibiotic cycloserine is dissolved in the minimum Volume of water (about a 25 percent solution). Ethanol is then slowly added to the concentrated solution. The crystalline antibiotic separates during this process.

The present application is a continuation-impart of our copending application Serial No. 216,254, filed March 17, 1951, and now abandoned.

As many apparently widely dierent embodiments of this invention may be made without departing from the spirit and scope hereof, it is to be understood that the above invention is not limited, except as dened in the appended claims.

What is claimed is:

1. A process for producing cycloserine which comprises cultivating strain No. ATCC 11924 of Sreptomyces Iavendulae in an aqueous, nutrient-containing solution under submerged aerobic conditions until substantial antibiotic activity is imparted to said solution.

2. A process :for producing cycloserine which comprises cultivating strain No. ATCC 11924 yof Streptomyces lavendulae in an aqueous, nutrient-containing carbohydrate solution under submerged aerobic conditions until substantial antibiotic activity is imparted to said solution, and then recovering -the so-produced cycloserine from the fermentation broth.

3. A process for producing cycloserine which comprises culti-vating strain No. ATCC 11924 of Streptomyces lavendulae in an aqueous, nutrient-containing `carbohydrate solution under submerged aerobic conditions at a temperature `of from about 25 to about 30 C. for -a period Iof from about two days to about four days and then recovering the so-produced cycloserine from the fermentation broth.

4. A process as claimed in claim 2 wherein the recovery of cycloserine includes the step of adsorbing the antibiotic on an ion-exchange resin containing sulfonic acid groups.

5. Cycloserine, an iamphoteric substance eective in inhibiting the growth of mycobacteria, which is highly soluble in Water and slightly soluble in methanol and which contains the elements carbon, hydrogen, nitrogen, and -oxygen in the following proportions:

Percent Carbon 35.3 Hydrogen 5.9 Nitrogen 27.4 Oxygen (by difference) 31.4

which has -a positive optical rotation in methanol and a suspension of which in mineral oil exhibits characteristic absorption in 4the infrared region of the spectrum at the following frequencies expressed in reciprocal centimeters: 1626, 1602, 1580, 1557, 1534, 1408, 1402, 1266, 1227, 1168, 1141, 1117, 1064, 938, 916, 892, 880, and 830.

References Cited in the le of this patent UNITED STATES PATENTS Foster June 17, 1947 Van Dolah Oct. 31, 1950 OTHER REFERENCES 

5. CYCLOSERINE, AN AMPHOTERIC SUBSTANCE EFFECTIVE IN INHIBITING THE GROWTH OF MYCOBACTERIA, WHICH IS HIGHLY SOLUBLE IN WATER AND SLIGHTLY SOLUBLE IN METHANOL AND WHICH CONTAINS THE ELEMENTS CARBON, HYDROGEN, NITROGEN, AND OXYGEN IN THE FOLLOWING PROPORTIONS: 